The present invention relates to methods and apparatus used to screen for specific binding interactions by fluorescent techniques. The methods are particularly useful for high throughput screening of such interactions in combinatorial libraries, including chip-based libraries, pin-based libraries, and split-pool libraries.
The development of techniques for the screening of large numbers of different molecules for possible biological activity has become important in developing new drugs. Known as xe2x80x9ccombinatorial chemistryxe2x80x9d techniques, these techniques typically involve the random generation of compounds to be screened. Combinatorial chemistry techniques are thus dramatically different from xe2x80x9crational drugxe2x80x9d design techniques, where specific structures for new compounds are designed based on a knowledge of other active compounds or the target site of the compound. Instead, combinatorial chemistry relies on the large numbers of candidate compounds generated, and the rapid screening of those compounds, to provide a sufficient probability of identifying active compounds.
Numerous different combinatorial chemistry techniques are known. For example, U.S. Pat. No. 5,445,934 to Fodor et al. describes a substrate in which large numbers of different polymers, such as nucleotides or peptides, are affixed to discreet regions of that substrate for the screening thereof for biological activity. In another example, U.S. Pat. No. 5,565,324 to Still et al. reviews xe2x80x9csplit-poolxe2x80x9d combinatorial libraries, and describes split pool libraries encoded with chemical tags.
The majority of work in developing new combinatorial chemistry techniques has focused on the methods for generating the combinatorial library, on the substrate structures for carrying the members of the library, or on the different structures from which the library can be comprised. Comparatively little attention has been devoted to developing new methods for detecting binding events within a combinatorial library. Accordingly, there is a need for new detecting methods that can be employed in conjunction with combinatorial chemistry techniques.
A method for detecting the binding of a test compound to a probe molecule comprises providing a test compound, the test compound having a first fluorophore bound thereto, and providing a screening substrate. The screening substrate comprises a solid support, a probe molecule bound to the solid support, and a second fluorophore bound to the solid support adjacent the probe molecule. An advantage of the invention is that this obviates the need for binding the second fluorophore directly to the probe molecule.
Preferably, the second fluorophore is bound to the solid support by means of a flexible linker group. This enables the second fluorophore to interogate different positions on the probe molecule, which is also bound to the solid support adjacent the linker group. As discussed in greater detail below, this enhances the ability of the method of the invention to detect positive binding events (specific binding of the test compound to the probe molecule).
The first and second fluorophores together comprise the donor and acceptor fluorophores of a fluorescence resonance energy transfer (FRET) pair, or a xe2x80x9cdonor/acceptor pair.xe2x80x9d In preferred embodiments, the first and second fluorophores are different, the first and second fluorophores emit light at different wavelengths from one another, and the second fluorophore absorbs light at a wavelength emitted by the first fluorophore.
The test compound is contacted to the screening substrate, and the screening substrate illuminated with light at a wavelength that is absorbed by the donor fluorophore (which may be either the first or second fluorophore). The transfer of energy from one to the other fluorophore (that is, from the donor to acceptor fluorophore) is then detected (for example, by detecting the presence or absence of light emitted by the acceptor fluorophore) with the transfer of energy indicating the binding of the test compound to the probe.
The method is useful in diagnostic assays for the detection of a particular compound, or in high through-put, or combinatorial, screening of multiple compounds. The method can be employed as a competition assay, where the test compound competes for binding with a target compound, where the binding of the target compound indicates the absence of the test compound.
Since the acceptor fluorophore is excited only when the donor fluorophore is in close proximity thereto, an advantage of the present invention is that there need not be a step of separating the test compound from the solid support (for example, an intervening washing step) to distinguish nonspecific binding from specific binding. For example, the entire process can be carried out in liquid, particularly aqueous, phase, by contacting a solution containing the test compound to the solid support, with the detecting step carried out while that solution is still in contact with the solid support.
The foregoing and other objects and aspects of the present invention are explained in detail below.